The development of mechanistically faithful models of congestive heart failure (CHF) biology is not readily feasible in cell culture or invertebrates as such systems fail to capture the complexity of multi- cellular or multi-organ processes. We have previously developed in vivo assays for complex multi- system phenotypes in the zebrafish, a screenable vertebrate, and exploited these in 'unbiased'screens for genetic and chemical modifiers. Adapting this integrated approach for CHF will allow us to identify the genes controlling CHF signaling hierarchies or small molecules that reverse CHF through novel mechanisms. These insights can be rapidly translated to other models for the dissection of CHF biology, and might also lead to novel diagnostic or therapeutic targets. We propose to develop a suite of core phenotypes and reporters for CHF in the zebrafish, and to combine these different elements in order to build a robust integrated in vivo assay for CHF in the following Specific Aims;Aim 1. To develop a range of independent phenotypes and reporters for CHF in the larval zebrafish Using mechanistically faithful zebrafish models of human CHF, we will develop scalable phenotyping tools and in vivo reporters for multiple components of the syndrome, specifically;contractility, cardiac output, vasomotor tone, as well as natriuretic peptide, renin-angiotensin and other myocardial or systemic pathways implicated in CHF. Aim 2. To define the optimal integrated zebrafish CHF assay for high-throughput screening We have recapitulated many human cardiovascular disease pathways in the larval zebrafish, at a stage when in vivo screening is feasible and high-resolution characterization of cardiac and vascular function is possible. We will validate multiple phenotypes and reporter lines in CHF models and test the feasibility of scaling to high-throughput while retaining specificity. We will adapt our optimized assays for automation in 96-well format, to generate an integrated assay with a robust z-factor for HTS. Aim 3. To perform a pilot chemical screen for heart failure modifiers A pilot screen of 7,500 of structurally-diverse small molecules will be performed in a zebrafish model of CHF to validate the assay for large-scale screening. 'Hits'will be further evaluated in high-resolution secondary assays, as well as in existing zebrafish morphants or mutants that perturb myocardial heart failure pathways. This work will facilitate 'omic scale approaches to the genetics and epigenetics of CHF, and will generate an array of small molecules for manipulating CHF pathways. These genes or pathway probes can be rapidly translated into other experimental systems, and small molecule hits may represent potential drug leads for the antecedents of CHF. Importantly, this combination of screen-mode in vivo physiology with chemical biology or genetics is generalizable to many other biologic problems.

Public Health Relevance

The biology of heart failure is extremely complex and involves interactions between the heart and many other organs including kidney, skeletal muscle and brain, as well as the immune system. Since these features cannot all be recreated in cell culture, we are using the tiny transparent zebrafish to develop models for the entire heart failure condition. These models can then be screened directly to find genes or environmental factors that influence the development of heart failure, and to identify new drugs for this common disease.